Enhanced indole biosynthesis

ABSTRACT

DNA molecules encoding a modified tryptophan synthase beta subunit are disclosed. When expressed in a recombinant host microorganism, these polypeptide analogs enable significant levels of intracellular indole production and accumulation. In the presence of an aromatic dioxygenase enzyme, the indole so produced can be converted to indoxyl, which upon exposure to air oxidizes to indigo.

This application is a division, of application Ser. No. 07/956,697, nowU.S. Pat. No. 5,374,543, filed Oct. 2, 1992 which is hereby incorporatedby reference.

FIELD OF THE INVENTION

The present invention relates to dye stuff biosynthesis bymicroorganisms, particularly the synthesis of indigo by bacteria. Thepresent invention describes an efficient, well regulated biosyntheticsystem wherein a precursor for microorganismic indigo production,indole, is produced intracellularly at high levels from glucose. Thisindole biosynthesis is mediated by an exogenous tryptophan operonmodified to promote indole production instead of tyrptophan synthesis.Indole produced in this manner can then be converted to indigo thoughthe action of another enzymatic system followed by exposure to air.Specifically, when the modified tryptophan operon taught herein isstably transformed into a microorganism harboring an appropriateadditional exogenous enzymatic pathway, indigo biosynthesis from glucoseoccurs when the microorganismic host strain is cultivated underappropriate conditions.

BACKGROUND OF THE INVENTION

The blue dye indigo is one of the oldest dyestuffs known to man. Its useas a textile dye dates back to at least 2,000 B.C. Until the late 1800s,indigo, or indigotin, was principally obtained from plants of the genusIndigofera, which range widely in Africa, Asia, the East Indies, andSouth America. As the industrial revolution swept through Europe andNorth America in the 1800s, demand for the dye's brilliant blue colorled to its development as one of the main articles of trade betweenEurope and the Far East. In 1883, Alfred yon Baeyer identified thechemical structure of indigo: C₁₆ H₁₀ N₂ O₂. In 1887, the firstcommercial chemical manufacturing process for indigo was developed, andis still in use today. This process involves the fusion of sodiumphenylglycinate in a mixture of caustic soda and sodamide to produceindoxyl. In the process's final step, indoxyl is then oxidized to indigoby exposure to air.

These commercial chemical processes for manufacturing indigo result notonly in production of the dye itself, but also in the generation ofsignificant quantities of toxic waste products. Obviously, a methodwhereby indigo may be produced without the generation of toxicbyproducts is desirable. One such environmentally sound method involvesindigo biosynthesis by microorganisms.

In a fortuitous discovery, Ensley et al. [(1983) Science, vol. 222, pp:167-69] found that a DNA fragment from a transmissible plasmid isolatedfrom the soil bacterium Pseudomonas putida enabled Escherichia colistably transformed with a plasmid harboring the fragment to synthesizeindigo in the presence of indole or tryptophan. Ensley et al. postulatedthat indole, added either as a media supplement or produced as a resultenzymatic tryptophan catabolism, was converted tocis-indole-2,3-dihydrodiol and indoxyl by the previously identifiedmulti-subunit enzyme napthalene dioxygenase (NDO) encoded by the P.putida DNA fragment. The indoxyl so produced was then oxidized to indigoupon exposure to air.

NDO had previously been found to catalyze the oxidation of the aromatichydrocarbon napthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronapthalene [Ensley et al., (1982) J. Bact.,vol. 149, pp: 948-54]. U.S. Pat. No. 4,520,103, hereby incorporated byreference, describes the microbial production of indigo from indole byan aromatic dioxygenase enzyme such as NDO. The NDO enzyme is comprisedof multiple subunits: a reductase polypeptide (Rd; molecular weight ofapproximately 37,000 daltons (37 kD)); an iron-sulfur ferredoxinpolypeptide (Fd; molecular weight of approximately 13 kD); and aterminal oxygenase iron-sulfur protein (ISP). ISP itself is comprised offour subunits having an α₂ β₂ subunit structure (approximate subunitmolecular weights: α, 55 kD; β, 21 kD). ISP is known to bind napthaleneand in the presence of NADH, Rd, Fd, and oxygen to reduce it tocis-napthalene-dihydrodiol. Fd is the rate-limiting polypeptide in thisnapthalene oxidation catalysis. See commonly assigned, allowed but notyet issued U.S. patent application Ser. No. 07/389,738, filed Aug. 4,1989, hereby incorporated by reference, for a thorough discussion of thevarious NDO subunits and ways to improve them for purposes of indigobiosynthesis.

In addition, aromatic dioxygenases other than NDO may also be useful inthe biosynthetic production of indigo. Ensley et al. also observed thata dioxygenase enzyme from another Pseudomonas strain capable ofdegrading toluene was also able to produce indigo when the culture mediawas supplemented with indole. For details, see U.S. Pat. No. 4,520,103,supra.

It has also long been known that microorganisms contain biosyntheticpathways for the production of all 20 essential amino acids, includingthe aromatic amino acid L-tryptophan. The de novo synthesis of aromaticamino acids (phenylalanine, tryptophan, and tyrosine) share a commonpathway up through the formation of chorismate. After chorismatesynthesis, specific pathways for each of the various aromatic aminoacids are employed to complete their synthesis.

Bacterial biosynthesis of tryptophan from chorismate is under thecontrol of the tryptophan (trp) operon. The trp operon, comprised ofregulatory regions and five structural genes, has been extensivelystudied because of its complex and coordinated regulatory systems. Theregulatory and structural organization of the trp operon, along with thecatalytic activities encoded by the structural genes of the operon,appear in FIG. 1. Of particular relevance to the present invention isthe conversion of indole-3'-glycerol-phosphate (InGP), in conjunctionwith L-serine, to L-tryptophan. The reaction is catalyzed by themulti-subunit enzyme tryptophan synthase (TS). During the reaction,indole is produced as an intermediate. However, the indole is veryrapidly combined with L-serine in a stoichiometric fashion to produceL-tryptophan. Thus, no free indole is produced as a result of this InGPplus L-serine conversion to tryptophan.

However, Yanofsky et al. (1959) Proc. Nat'l. Acad. Sci. Vol. 45, pp.1016-1026, identified a tryptophan synthase mutant which lead to theaccumulation of indole. This particular mutant, however, was subject tospontaneous reversion to the wild-type phenotype, as the mutationresulted from a single nucleotide base pair change in a gene coding forone of subunits of tryptophan synthase.

Thus, the goal of the present invention was to create stable tryptophansynthase mutants capable of yielding high levels of intracellularindole. When such indole accumulating routants also express an aromaticdioxygenase enzyme like NDO, this accumulated indole may be converted toindoxyl. Indoxyl so produced may then oxidize to indigo upon exposure toair. Through the commercial application of recombinant DNA technology, anovel and environmentally sound biosynthetic indigo production methodhas been developed utilizing microorganisms stably transformed withexogenous DNA molecules encoding a modified trp operon and an aromaticdioxygenase enzyme.

DEFINITION OF TERMS

The following terms will be understood as defined herein unlessotherwise stated. Such definitions include without recitation thosemeanings associated with these terms known to those skilled in the art.

A trp operon useful in securing microorganismic indole accumulation is atrp operon, isolated from a microorganism as a purified DNA moleculethat encodes an enzymatic pathway capable of directing the biosynthesisof L-tryptophan from chorismate. Indole accumulation is enabled bymodification of one or more of the pathway's structural elements and/orregulatory regions. This modified trp operon may then be introduced intoa suitable host microorganism. It should be noted that the term "indoleaccumulation" does not necessarily indicate that indole actuallyaccumulates intracellularly. Instead, this term can indicate that indoleis produced and made available as a substrate for intracellularcatalytic reactions other than the formation of L-tryptophan. In thecontext of this invention, the "accumulated" indole may be consumed inthe conversion of indole to indoxyl by an aromatic dioxygenase such asNDO, or it may actually build up intracellularly, as would be the casewhen the desired end product is indole.

As used herein, "enhanced" indole accumulation refers to theintracellular production and/or accumulation of indole beyond thatobserved in the mutant identified by Yanofsky et al., supra, namely whenasparagine is substituted for lysine at amino acid position 382 of thetryptophan synthase beta subunit polypeptide, or by a recombinantmicroorganism coding for that same mutation. The determination ofwhether enhanced indole accumulation occurred involves a comparison ofindole accumulation due to new analogs in contrast to the indoleaccumulated under the same conditions by the analog having asparagine atamino acid position 382 of the tryptophan synthase beta subunit.

A suitable host microorganism is an autonomous single-celled organismuseful for microbial indole and/or indigo production and includes botheucaryotic and procaryotic microorganisms. Useful eucaryotes includeorganisms like yeast and fungi. Prokaryotes useful in the presentinvention include bacteria such as E. coli, P. putida, and Salmonellatryhimurium.

Bi-synthetic conversion of indole to indigo is meant to include indoxyloxidation to indigo mediated by air.

A DNA molecule used herein may encode regulatory and/or structuralgenetic information. A DNA molecule according to the instant inventionshall also include: nucleic acid molecules encoding sequencescomplementary to those provided; nucleic acid molecules (DNA or RNA)which hybridize under stringent conditions to those molecules that areprovided; or those nucleic acid molecules that, but for the degeneracyof the genetic code, would hybridize to the molecules provided or theircomplementary strands. "Stringent" hybridization conditions are thosethat minimize formation of double stranded nucleic acid hybrids fromnon-complementary or mismatched single stranded nucleic acids. Inaddition, hybridization stringency may be effected by the variouscomponents of the hybridization reaction, including salt concentration,the presence or absence of formamide, the nucleotide composition of thenucleic acid molecules, etc. The nucleic acid molecules useful in thepresent invention may be either naturally derived or synthetic.

An "exogenous" DNA molecule is one that has been introduced into thehost microorganism by a process such as transformation, transfection,conjugation, electroporation, etc. Please note that it is possible thatthe host cell into which the "exogenous" DNA molecule has been insertedmay itself also naturally harbor molecules encoding the same or similarsequences. For example, when E. coli is used in this invention as thehost strain, it is recognized that normally the host naturally contains,on its chromosome, a trp operon capable of directing the synthesis ofL-tryptophan from chorismate under conditions enabling trp operonexpression. A molecule such as this is referred to as an "endogenous"DNA molecule.

A stably transformed microorganism is one that has had one or moreexogenous DNA molecules introduced such that the introduced moleculesare properly maintained, replicated, and segregated. Stabletransformation may occur by chromosomal integration or byextrachromosomal element, such as a plasmid vector. A plasmid vector iscapable of directing the expression of polypeptides encoded byparticular DNA molecules. Expression is regulated by an inducible (orrepressible) promoter that enables high levels of transcription offunctionally associated DNA molecules encoding specific polypeptides,such as the structural genes of a trp operon modified as describedherein.

The following three-letter abbreviations for the 20 essential amino acidresidues are used throughout the specification: Ala (Alanine), Arg(Arginine), Asn (Asparagine), Asp (Aspartic acid), Cys (Cysteine), Glu(Glutamic acid), Gln (Glutamine), Gly (Glycine), His (Histidine), Ile(Isoleucine), Leu (Leucine), Lys (Lysine), Met (Methionine), Phe(Phenylalanine), Pro (Proline), Set (Serine), Thr (Threonine), Trp(Tryptophan), Tyr. (Tyrosine), and Val (Valine).

SUMMARY OF THE INVENTION

One aspect of the present invention is to provide DNA molecules encodingpolypeptide analogs of a tryptophan synthase beta subunit. When suchanalogs are incorporated into tryptophan synthase, indole accumulatesintracellularly at levels in excess of that observed when lysine isreplaced by asparagine at amino acid position 382 of the beta subunit.Typically, the tryptophan synthase beta subunit analogs are encoded byDNA molecules wherein at least one codon corresponding to a specificamino acid position in the DNA molecule's expression product has beensubstituted for another codon. Particularly useful codon substitutionscan be made at the codons corresponding to amino acid positions trpB³⁷⁹and trpB³⁸². At the codon corresponding to the amino acid positiontrpB³⁷⁹, those codons that can be substituted include those coding forVal, Ile, Leu, Ala, and particularly Pro. In contrast, useful codonsubstitutions at the codon corresponding to the amino acid positiontrpB³⁸² include those coding for Gly and particularly for Met. DNAmolecules comprising codon substitutions at both codons corresponding toamino acid positions trpB³⁷⁹ and trpB³⁸² also result in the productionof enhanced amounts of intracellular indole, particularly when the codoncorresponding to amino acid position trpB³⁷⁹ codes for Pro and the codoncorresponding to amino acid position trpB³⁸² codes for Met.

Another aspect of the invention provides for a tryptophan synthase betasubunit analog which, when assembled into tryptophan synthase, resultsin enhanced indole accumulation relative to a tryptophan synthasecomprising a tryptophan synthase beta subunit Asn³⁸² analog. In oneembodiment, the tryptophan synthase beta subunit analog comprises asubstitution of one amino acid residue for another at one or more aminoacid positions in the natural tryptophan synthase beta subunit aminoacid sequence. Particularly useful amino acid residue substitutionsinclude those at amino acid positions 379 and 382 of the naturaltryptophan synthase beta subunit amino acid sequence. Amino acid residuesubstitutions at amino acid position 379 include Val, Ile, Leu, Ala, andparticularly Pro in place of Arg, while at amino acid position 382, Glyand particularly Met can be substituted for Lys. In a preferredembodiment of the tryptophan synthase beta subunit analog, Pro issubstituted for Arg at amino acid position 379 and Met is substitutedfor Lys at amino acid position 382.

A further aspect of the invention is the stable transformation ortransfection of a procaryotic or eucaryotic host cell with the DNAmolecules taught herein in a manner allowing the host cell to expressthe encoded tryptophan synthase beta subunit under appropriateconditions. The procaryotic host Escherichia coli represents one suchpreferred host microorganism.

A biologically functional plasmid or viral DNA vector including a DNAmolecule of the invention represents another aspect of this invention.In one embodiment, a eucaryotic or procaryotic host cell, such as E.coli, is stably transformed or transfected with such a biologicallyfunctional vector.

Other aspects of the invention involve methods for the biosynthesis ofindole and indigo using the DNA molecules of the invention.Microorganismic indole production can be accomplished by stablytransforming or transfecting a host microorganism with a DNA molecule ofthe invention and cultivating the microorganism under conditionsenabling the biosynthesis of indole. Similarly, indigo can be producedby further transformation or transfection of the above microorganismwith a DNA molecule encoding an aromatic dioxygenase enzyme, such asnapthalene dioxygenase. Cultivating the microorganism under conditionsfacilitating the expression of the DNA molecules encoding the tryptophansynthase beta subunit analog and the aromatic dioxygenase enablesintracellular indole accumulation and conversion of indole to indoxyl,which is then oxidized to indigo by exposure to air.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a physical map identifying the various regulatory andstructural elements of the E. coli trp operon. Additionally, the variousproteins encoded by the structural genes and the chemical reactionscatalyzed thereby are also described.

FIG. 2 graphically illustrates indole synthesis and accumulation duringa 1L fermentation of pYTrp#26.

FIG. 3 graphically depicts 1L fermentations of mutant 5 [(--□--),without anthranilate; (--▪--) with 300 mg/L anthranilate] and pYTrp#26[(--♦--), with anthranilate; (--⋄--), without anthranilate].

Numerous aspects and advantages of the invention will be apparent tothose skilled in the art upon consideration of the following detaileddescription which provides illumination of the practice of the inventionin its preferred embodiments.

DETAILED DESCRIPTION

Present methods of biosynthetic indigo production employ only thebioconversion of indole to indigo utilizing an aromatic dioxygenase likeNDO. This necessitates the addition of indole to the culture media, asno intracellular indole accumulation occurs in such systems. However,indole added to the culture media may be toxic to microorganisms. E.coli growth may be inhibited when indole is present in the media. Banget al. [(1983) Biotechnology and Bioengineering, vol. 25, pp: 999-1011]described the effects of adding exogenous indole to E. coli being grownin shake flasks in minimal media. They found that while concentrationsof up to 0.025% slowed bacterial growth, the cells acclimated to thepresence of indole over time. However, 0.03% indole severely limitedgrowth with no apparent acclimation, and indole concentrations above0.04% prohibited growth altogether. In addition, Bang et al., supra,found that L-tryptophan synthesis was inhibited when indole was added atconcentrations in excess of 0.2 g/100 ml.

To avoid the inherent limitations of indigo synthesis through indolemedia supplementation, a system capable of endogenous indolebiosynthesis is required. One such system may employ transferring anexogenous DNA molecule encoding a DNA sequence for a trp operon,modified so as to promote indole production and accumulation, into arecombinant host microorganism already capable of expressing NDO(preferably altered as discussed above). Such a system would allow forthe production of indigo from glucose or other carbon sources.Optimally, such a system would efficiently convert the endogenouslyproduced indole to indoxyl in a manner avoiding intracellular indoleaccumulation.

It has long been known that indole is produced as an intermediate inL-tryptophan biosynthesis. However, the indole so produced exists onlytransiently, i.e. it exists only during the bioconversion of InGP andL-serine to L-tryptophan. No soluble free indole accumulates as a resultof this bioconversion, which is catalyzed by the multi-subunit enzymetryptophan synthase (TS). TS is one of several enzymes encoded by thetrp operon and has been extensively studied. For example, see Hyde etal., (1990) Biotechnology, vol. 8, pp: 27-32; Djavadi-Ohaniance et al. ,(1986) Biochemistry, vol. 25, pp: 2502-08; and Ahmed et al., (1986)Biochemistry, vol. 25, pp: 3118-24. The reactions catalyzed by the fivegene products encoded by the trp operon are depicted in FIG. 1.

TS is comprised of an α₂ β₂ subunit structure. The α subunit catalyzesthe conversion of InGP to indole and D-glyceraldehyde 3'-phosphate andhas an approximate molecular weight of 29 kD. The β subunit, which hasan approximate molecular weight of 43 kD and catalyzes the reactionL-serine plus indole to make L-tryptophan, liberating one molecule of H₂O in the process. The β subunits exist as a dimer, called the β₂subunit. β₂ associates with two α subunits to form the α₂ β₂ TSholoenzyme which has an extended αββα quaternary structure. Theholoenzyme catalyses the reaction of L-serine plus InGP to produceL-tryptophan, D-glyceraldehyde 3'-phosphate, and one molecule of H₂ O.No free indole is produced because it is a "channeled" intermediate,i.e. indole produced at the α subunit active site is intramolecularlytransferred by internal diffusion to the β subunit active site [Hyde etal., (1990) supra]. TS mutants unable to properly channel indole fromthe α subunit active site to the β subunit active site, or that have analtered β subunit active site, may be unable to combine L-serine withindole and thus may be useful in indigo biosynthesis as free solubleindole may be provided upon which NDO can act.

The DNA sequence of the E. coli trp operon was published in 1981 byYanofsky et al. [Nucl. Acids Res., vol. 9, no. 24, pp:6647-6668]. Thefive structural genes of operon are transcribed as one polycistronicmessage of about 6,800 ribonucleotides in length. The 5'-most gene,trpE, is encoded by nucleotides 279-1841 of this polycistronic messengerRNA (mRNA). Nucleotides 1841-3436 code for the trpD gene product, whiletrpC is coded for by nucleotides 3440 to 4798. Because TS is the enzymeknown to produce and then utilize indole in the production oftryptophan, the genes encoding the α and β subunits, namely the trpA(mRNA nucleotides 6003-6809) and trpB (mRNA nucleotides 4810-6003)genes, respectively, may be subcloned into an appropriate vector so thatsite directed mutagenesis may be conducted so as to render the resultantTS holoenzyme incapable of combining indole with serine to producetryptophan.

As previously noted, a point mutation near the carboxy terminus(C-terminus) of the trpB gene, [SEQ ID Nos: 4 and B] specifically atamino acid position 382, was observed by Yanofsky et al., supra, (1958)to lead to intracellular indole accumulation. Later, this singlenucleotide change was found to have occurred at the third nucleotideposition in the codon normally coding for lysine. This mutation resultedin asparagine being substituted for lysine at this position. Because ofthis mutation's third position character and deleterious effect on thecell's ability to synthesize tryptophan, it was very unstable andsubject to reversion to the wild type genotype. Accordingly, to avoidspontaneous reversion to the wild type sequence, any engineered mutationwill preferably involve more than one nucleotide base pair changewhenever possible.

The widely known technique of site directed mutagenesis provides a readymechanism whereby the lysine to asparagine change at the codoncorresponding to amino acid position 382, designated as Lys³⁸² toAsn³⁸², of the trpB gene can be stabilized. One such stabilized forminvolves the generation of a double mutant at this particular codon,corresponding to trpB amino acid position 382 (trpB382), thuseffectively preventing spontaneous reversion to the wild type genotypeand phenotype. Additionally, as a substitution at this particular trpBresidue was observed to lead to intracellular indole accumulation, otheramino acids may also be substituted at this position in an effort toimprove or enhance indole accumulation. For example, substitutions maybe made based on differences in side chain charge or size. One suchpreferred change involves the substitution of glycine for lysine attrpB³⁸². In another preferred embodiment, methionine may be substitutedfor lysine at this position, although this particular substitution,while leading to greater indole accumulation than the Asn³⁸²substitution of Yanofsky et al., supra, (1958) produces less indole thanthe Gly³⁸² mutation.

Amino acid substitutions at positions other than trpB³⁸² may also proveuseful in generating indole-accumulating mutants. For instance, aminoacid substitution at trpB³⁷⁹, represented by arginine in the wild type βsubunit, may lead to an even more significant level of indoleaccumulation. Useful amino acid substitutions at trpB³⁷⁹ may includereplacing the wild type residue with Ala, Ile, Leu, Pro, or Val, inaddition to other amino acid residues. In fact, when proline issubstituted for arginine at trpB³⁷⁹ indole is seen to accumulateintracellularly to a level 20 times that found for the Yanofsky et al.mutation. It is likely that other amino acid positions in trpB can alsobe mutagenized favorably with respect to indole accumulation. Otherpotential useful changes may include disrupting the indole "channel,"thus preventing the movement of indole from the α subunit to the βsubunit of the tryptophan synthase holoenzyme. Likewise, one or moreamino acid substitutions at residues believed to be involved in theconversion of indole and L-serine to L-tryptophan in the β subunit arealso possible. See Hyde et al., supra, (1990) .

It should be noted that in addition to amino acid substitutions atparticular amino acid residues, the present invention also envisions theinsertion of additional amino acid residues at one or more particularpositions as well as the deletion of one or more specific residues. Inaddition, combinations of various useful mutations causing increasedindole accumulation, such as amino acid substitutions, insertions,and/or deletions, also fall within the scope of this invention. One suchdouble mutant, designated pYTrp#26, incorporates amino acidsubstitutions at two different positions. Specifically, pYTrp#26represents the following changes from the wild type β subunit: Arg³⁷⁹was changed to Pro³⁷⁹ and methionine was substituted for lysine attrpB³⁸².

Beyond the above trpB mutations, other mutations within the trp operonmay also prove useful in enabling microorganismic indole accumulation.Of particular interest are mutations in the trpA gene, which uponexpression may still result in a subunit capable of assembly into the TSholoenzyme and catalyzing the conversion of InGP to indole andD-glyceraldehyde-3'-phosphate, but being incapable of participating inthe requisite indole "channeling" required for L-tryptophan synthesis.Also, because the β subunits of the TS holoenzyme comprise approximatelytwo-thirds of the indole "channel," Hyde et al., supra, (1990)]mutations which deleteriously affect this region's ability to facilitateindole "channeling" are also envisioned by the present invention.Further, site-directed mutagenesis may be used to engineer amino acidsubstitutions, deletions, and/or insertions in the active site of the βsubunits. For example, an amino acid substitution at Lys⁸⁷ in the βsubunit may produce a β subunit still capable of assembling into the TSholoenzyme, but that is incapable of catalyzing the bioconversion ofL-serine and indole to L-tryptophan.

In addition to making specific amino acid changes in the variouspolypeptides encoded by the genes of the trp operon, site-directedmutagenesis may be employed to alter the structural organizationaland/or regulatory regions of the trp operon. The operon's regulatorysystems are complex and coordinated, being comprised of at least threelevels of regulation. At the protein level, in the presence of excessL-tryptophan, anthranilate synthase, a multi-subunit enzyme whosesubunits are encoded by the trpE and trpD genes, experiences feedbackinhibition [Henderson et al., (1970) J. Biol. Chem., vol. 245,pp:1416-1423]. At the transcriptional level, in the presence of excessL-tryptophan, activated trp repressor molecules limit transcriptioninitiation to about 1% of its maximal rate. Under these conditions,attenuation (for an explanation, see Yanofsky, C. (1987) TIG, vol. 3,no. 12, pp: 356-360; Yanofsky et al., (1981) supra), involving bothtranscriptional and translational regulation, can suppress structuralgene transcription another six-fold, although a significant basal levelof trp operon expression still occurs when excess L-tryptophan ispresent [Roesser et al., (1989) J. Biol. Chem., vol. 264, no. 21, pp:2284-2288]. Thus, mechanisms enabling more stringent transcriptionaland/or translational control may be useful with respect to indole andindigo biosynthesis.

Removal of the endogenous trp promoter from plasmid constructionsharboring the trp operon may provide for enhanced regulatory control. Inone embodiment, the 7.4 kb Eco RI - Sal I fragment encoding the trpoperon may be cloned into the expression vector pAC1, the constructionof which is described in detail in U.S. patent application Ser. No.07/389,738, supra. This construction is designated pYTrp. The pAC1plasmid vector employs the heat-inducible phage lambda P_(L) promoter todirect the expression of DNA sequences inserted proximately downstream.Please note that expression systems employing the P_(L) promoter requirethe presence of the repressor protein mutant cI857 for appropriateregulation. The low-level, constitutively expressed gene encoding cI857may be inserted into the chromosome of an appropriate host strain, suchas E. coli strain FM5 (A.T.C.C. accession no. 53911) or it may beplasmid borne.

Constructions such as pYTrp may be modified to include the variousmutants alluded to in this invention. In one embodiment, designatedpYTrp#26, a DNA fragment encoding a modified trpB gene encoding apolypeptide capable of producing elevated levels of intracellular indolewas subcloned into pYTrp after excision of the wild type sequence. Whencultivated in a 1 L (liter) fermentor, pYTrp#26 produced about 30% ofits total yield of indole prior to PL temperature induct ion. When theendogenous trp promoter was removed from the pYTrp#26 construct byremoving about 400 bp (base pairs) of upstream, noncoding trp operonDNA, the new construction, pYTrp#26p-, showed considerably tighterregulation and enhanced host cell growth as compared to a bacterialstrain harboring pYTrp#26.

However, the pYTrp#26p- construct still contained the trp attenuatorregion, which may be responsible for up to 90% of trp operon repressionin vivo. In a preferred embodiment, a plasmid, designated pYTrp#26att-,was constructed in which both the trp promoter and attenuator regionsare deleted from the exogenous trp operon construct. Thus, thestructural genes for the indole-producing trp operon construction can beregulated solely by the promoter designed to drive the expression ofinserted heterologous DNA sequences when such sequences are cloned intothe expression vector. To further optimize expression of the insertedtrp structural genes, a strong ribosomal binding site with consensusspacing can be inserted between the expression vector's promoter, suchas the P_(L), and the 5' end of the trpE gene.

Plasmids other than pAC1 may also be useful in practicing the presentinvention. The entire trp operon, or one of the preferred variantstaught herein, may be inserted into a plasmid such as pBR322. In oneembodiment, a construction designated pBRYTrp (containing the entire trpoperon from pYTrp#26p-) was generated. Such constructions may exhibitenhanced, or in the case of pBRYTrp#26p-, reduced expression control ofthe inserted DNA sequence(s). Reduced control of expression (prior tothermal induction) in cases where high copy number plasmids such aspBR322 are used in conjunction with the P_(L) promoter is perhaps due totitrating out the small number of cI857 repressor molecules present inthe host cell. Accordingly, when moderate to high copy number plasmidsare used in accordance with this invention, externally regulatedpromoters other than P_(L) may prove useful.

Another modification one may make to an indole-producing trp operon isto delete, in whole or in part, the 3' untranslated portion of theoperon. In E. coli, as well as in the 7.4 kb fragment used as thestarting material in this invention, this 3' region contains both arho-dependent and rho-independent transcription termination sequence.Removal of either or both of these sequences may be readily accomplishedby those skilled in the art. In one embodiment of the present invention,only the rho-dependent terminator is eliminated, deleting approximately250 bp 3' of the trpA gene. However, should both termination sequencesbe removed, it is preferable that another termination sequence presentin the vector DNA be functionally associated with the 3' end of theinserted trp operon to insure efficient transcription termination.

Other useful modifications of a trp operon useful in the practice of thepresent invention may also be made. For example, in the naturally E.coli trp operon, it has been observed that the initiator codon of thetrpD and trpA genes overlap with the termination codons of the trpE andtrpB genes, respectively. The DNA sequence, 5 '-TGATG-3', involved inthese overlaps is identical. Yanofsky et al., supra, (1981). Thisoverlap has been referred to as "translational coupling" and is perhapsan evolutionarily evolved device used in the translation ofpolycistronic maNAs to ensure proportionate production of functionallyrelated polypeptides or equimolar production of proteins that areconstituents of a multi-enzyme complex. Das et al., (1984) Nucl. AcidRes., vol. 12, no. 11, pp: 4757-4768. The translation products of boththe trpE/trpD and trpB/trpA messages form αββα complexes. In contrast,the trpC gene, the only member of this operon which does not code for apolypeptide incorporated into a multi-subunit enzyme, does not overlapwith either the trpD gene terminator or the trpB initiation sequences.Instead, the trpC gene is flanked by six untranslated nucleotides at its5' end and 14 untranslated nucleotides at its 3' end.

It may be desirable to eliminate the overlaps present in the trpE/trpDand trpB/trpA sequences. This may be accomplished by manufacturing asmall synthetic double stranded DNA fragment spanning two restrictionsites or by site directed mutagenesis. Using either approach, theinserted DNA sequence may be designed to physically separate thetermination and initiation codons of the various genes. The sequenceintervening between the termination and initiation codons may merelyserve as a spacing sequence. However, Das et al., supra, suggest mereseparation of translationally coupled messages can lead to decreasedlevels of the translation products. Thus, any separation performed onthese overlapping sequences should not be for mere spacing. Instead,such sequences should be designed so that upon termination oftranslation of the more 5' message, translation initiation of the moredistal gene by the same ribosome is possible. Thus, a ribosomal bindingsite, and/or one or more restriction enzyme recognition sequences toimprove the clonability of the various trp operon genes, might beincluded in such a spacer region. Additionally, the untranslated regionsflanking the trpC gene could also be modified to include one or more ofthe above mentioned possibilities.

It is known that the trp operon contains at least six ribosomal bindingsites, also known as Shine-Delgarno sequences [Yanofsky et al., supra,(1981) ]. Each of the five trp structural genes and the leader sequence(comprising the attenuator) are each preceded by such a sequence.However, in the case of trpD, trpC, trpB, and trpA, the Shine-Delgarnosequence is located within the coding region of the gene immediatelypreceding it. Thus, in an effort to optimize translation efficiency, assuggested in the previous paragraph, it may be preferable to modify theoperon such that each gene be sufficiently removed from the others so asto allow for ribosomal binding sites in untranslated regions immediatelyadjacent to the 5' end of each gene. Such modifications could beconducted for any one, some, or all of the five trp structural genes,although in the cases of the trpE/trpD and trpB/trpA genes, attentionwould have to be directed at maintaining termination and initiationcodons.

The trp operon is de-repressed in E. coli only when the organismencounters an environment depleted or devoid of tryptophan, although acontinuous low level of expression is observed at all times in order torespond to environmental stress. As a result of this only intermittentde-repression, the codon usage observed in the coding regions of the trpstructural genes is characteristic of that seen in other moderatelyexpressed E. coli genes, namely it is non-random but less restrictedthan the codon usage observed in highly expressed E. coli genes. Becausethe goal of this invention is the development of an efficient,commercially viable biosynthetic indigo production system, it may bepreferred to enable a rate of indole synthesis in excess of that whichis possible using a modified, albeit mostly natural, trp operon. Alongthese lines, one may construct a trp operon that incorporates, in wholeor in part, only codons found in highly expressed E. coli proteins.These codons, often called "preferred" codons, are widely known in theart. To generate such an optimized operon, or a part thereof, one couldemploy the procedures described by Stabinsky, U.S. Pat. No. 4,897,471,hereby incorporated by reference. Should a host other than E. coli beused, such as a yeast or other bacterial strain, it would be desirableto utilize codons preferred by that organism in constructing a trpoperon in accordance with this invention.

All major microbial groups possess the ability to synthesize tryptophanunder appropriate conditions. All enteric bacterial species appear toharbor trp operons structurally organized as that found in E. coli.Other types of bacteria have the genes encoding the various trp operoncomponents at various chromosomal locations. The elements of suchdispersed systems may be independently regulated as well. In addition,the various components of the trp operons in such microorganismspotentially contain genes coding for polypeptides having varying degreesof amino acid homology when compared to their counterparts in E. coli.For example, the trpA gene found in the close E. coli relative S.typhimurium is 85% homologous at the nucleotide level and 96% homologousat the amino acid level .when compared to the trpA gene and protein fromE. coli. It is likely that greater differences exist with othermicroorganisms that are more distantly related to E. coli. Thus, withinthe scope of this invention the possibility exists that a highlyefficient hybrid trp operon could be constructed incorporatingcomponents from various microorganismic trp operons and that when such ahybrid operon is modified as is taught herein, it could be moreefficient in the high level production of indole than any modified butunhybridized trp operon.

Because microorganismic indigo biosynthesis, absent indole-supplementedmedia, requires that the microorganism be able to synthesize exogenousenzymatic pathways capable both of producing indole and then catalyzingindole's conversion to indoxyl, various combinations of plasmidsencoding these different pathways may be utilized. To insure propermaintenance, segregation, and propagation of an indigo producing systememploying more than one plasmid, the plasmids used must be fromdifferent complementation groups. In addition, transcription of thegenes of the two pathways may be under the control of a single type ofpromoter, such that upon induction, both pathways are transcribed. Forexample, in a two plasmid system, expression of both pathways may beunder the control of the P_(L) promoter or a suitable alternative.Alternatively, each pathway may be under the control of a promoterinduced by a different mechanism. Such a system would enable theinduction of the two pathways at different times, if desired. In thisway, indole accumulation could begin prior to NDO synthesis.Alternatively, NDO could be synthesized prior to transcription andtranslation of the modified trp operon, perhaps to enable the conversionof indole to indigo without allowing intracellular indole to accumulateto toxic levels prior to its bioconversion to indoxyl.

Also within the scope of the invention is the generation of a singleplasmid system upon which both enzymatic pathways are harbored. Again,in such a system, each pathway may employ a promoter of the same type,thus enabling simultaneous expression of both operons. However, it isalso possible that each pathway could employ a promoter inducible byindependent mechanisms, thus enabling the induction of each pathwaysimultaneously or at different times. In yet another aspect, bothpathways may be functionally associated so that only one promoter needbe employed. This single promoter would enable transcription of bothoperons. The operons could be arranged so that the NDO pathway isadjacent to the regulatable promoter, followed by the trp operon.Likewise, the trp operon could be inserted before the NDO operon.

Additionally, it is possible to develop microorganismic indole and/orindigo producing systems wherein one or more of the genes encodingpolypeptides involved in these processes are integrated into thechromosome of the host microorganism, as opposed to being located on oneor more extrachromosomal elements. Rapid and irreversible chromosomalintegration can be directed by an integration plasmid designed todeliver into the host microorganism's chromosome (via recombination)cloned DNA molecules. Such integration plasmids, containing the DNAmolecules intended to be integrated, are transformed into the desiredhost microorganism. Such plasmids are capable of maintenance,propagation, segregation, and copy number control. In addition,selectable markers, such as one or more drug resistance genes, may beincluded. Essential is the inclusion of integration sequences capable ofdirecting the translocation event. Such integration sequences may beobtained from a variety of bacteriophage and plasmid sources. The DNAmolecules intended to be translocated will comprise, in addition to thedesired structural gene(s), the requisite regulatory genes and/orelements required for proper expression regulation of the includedstructural genes. Once integrated, the trp operon gene(s) and/oraromatic dioxygenase encoding molecule(s) may be expressed underappropriate conditions, thereby facilitating intracellular indoleproduction and/or indigo biosynthesis.

The general recombinant DNA techniques used in the present invention,like DNA isolation and purification, cleavage of DNA with restrictionenzymes, construction of recombinant plasmids, introduction of DNA intomicroorganisms, and site directed mutagenesis, are described in manypublications, including Mannjarls et al., Molecular Cloning--ALaboratory Manual, Cold Spring Harbor Laboratory (1982) and CurrentProtocols in Molecular Biology, edited by Ausubel et al., GreenePublishing Associates and Wiley Interscience (1987).

The following examples are offered to more fully illustrate the presentinvention. In addition, the Examples provide preferred embodiments ofthe present invention but are not meant to limit the scope thereof.

EXAMPLE 1 Trp Operon Cloning

The 7.4 kb fragment encoding the entire trp operon was excised fromplasmid pGX50 (NRRL B-12264) using Eco RI to Sal I. Following agarosegel purification, this fragment was then ligated into pAC1 which hadpreviously been digested with Eco RI and Xho I and then phosphatased toprevent reannealing of vector and polylinker. The resultant plasmidconstruct was designated pYTrp.

EXAMPLE 2 Generation of trpB Indole-accumulating Mutants

To generate a trp operon capable of directing the high levelaccumulation of intracellular indole, pYTrp was then digested with Hpa Iand Bam HI, releasing a 1.2 kb fragment containing the C-terminal regionof the trpB gene in conjunction with the entire trpA gene. Followingagarose gel purification of the Hpa I/Bam HI fragment, it was ligatedinto plasmid p1036 which had been previously digested with Hpa I and BamHI. p1036 was generated by substituting the Sst I to Aat II fragment(containing a Kanamycin resistance gene) of pCFM836 (see U.S. Pat. No.4,710,473, hereby incorporated by reference) with a similar fragmentfrom pCFM636 (U.S. Pat. No. 4,710,473, supra) and by substituting theDNA sequence between the unique Aat II and Eco RI (containing asynthetic P_(L) promoter) restriction sites with the followingoligonucleotide duplex: ##STR1## Cloning of the Hpa I/Bam HI fragmentgenerated the intermediate plasmid p1036A/B. p1036A/B was then digestedwith Eco RI and Bam HI and the 1,200 bp fragment carrying the desiredsequence was gel purified. The resultant gel-purified fragment was thenligated into similarly digested M13mpll RF DNA and transformed intocompetent E. coli JM109 by standard techniques. A plaque found tocontain the desired construct was isolated and designated mpA/B. Singlestranded (SS) DNA was then prepared from mpA/B to serve as the substratefor site-directed mutagenesis according to standard procedures.

As it was known that a particular trpB point mutation at the codoncorresponding to amino acid position 382 (whereby Asn was substitutedfor Lys) had previously been observed to result in the production ofdetectable levels of intracellular indole [Yanofsky et al., supra,(1958),], oligonucleotides were designed and synthesized to enable thesubstitution of the wild type residue for another at this position. Asthe DNA sequence of the trp operon, and the trpB gene in particular, hasbeen described in the literature, the design of oligonucleotides usefulherein is readily within the skill of the art. The trpB DNA sequence[SEQ ID NO:3], and the corresponding amino acid sequence, that is ofparticular relevance to the present invention is as follows: ##STR2##Each engineered substitution was designed to alter at least twocontiguous nucleotides, thus substantially reducing the likelihood of areversion to the wild type genotype. With these oligonucleotides inhand, site-directed mutagenesis was then conducted using SS mpA/B DNA.

Following the mutagenesis reactions, the products were serially dilutedand transformed into competent JM109 and plated. Following an overnightincubation, plates containing several hundred plaques for each of thevarious mutations were overlaid with nitrocellulose, allowing phageparticles to be transferred. Following denaturation and neutralization,the filters, to which the SS phage DNA was now bound, were baked underhouse vacuum for 2 hours at 80° C. The filters were then hybridized toradiolabelled probes as described in Manniatis et al., supra, the probebeing that oligonucleotide which had been used for that specificmutagenesis reaction. Following hybridization, the filters were washedunder stringent conditions [2X SSC (1X SSC=0.15M NaCl, 0.015M sodiumcitrate, pH 7.0), 1% sodium dodecyl sulfate (SDS), 4° C. below thetheoretical melting temperature] to remove non-specific hybridizationand then autoradiographed. The washing conditions used varied due to theuse of probes different of different lengths and of different sequences.For the purposes of these hybridizations and washings, oligonucleotidemelting temperatures (T_(M)) were calculated by allocating 2° C. foreach A or T in the probe and 4° C. for each C or G, and then summing theresult for each oligonucleotide.

Using the autoradiography results, putative positive plaques wereisolated and subjected to at least one round of plaque purification. Oneor more of those plaques found to strongly hybridize to its specificprobe was removed from its corresponding plate, serially diluted, andused to transfect a fresh JM109 culture in logarithmic phase. Followinga brief infection period, the mixtures were then plated and allowed togrow out. The nitrocellulose binding and hybridization procedure wasthen conducted again for each putative mutant.

Upon confirmation by hybridization that the desired mutant had beenobtained, RF DNA for each mutant was prepared. This RF DNA was thendigested with Hpa I and Bam HI, thus excising a particular trpB mutantas a Hpa I to Bam HI fragment. For each mutant, this fragment could thenbe ligated into pYTrp which had previously been digested with Hpa I andBam HI and gel purified. These ligation reactions were then used totransform competent E. coli strain FM5. A colony of each of theresultant transformants was then cultivated in a shaker flask underconditions allowing for the transcription and translation of theplasmid-borne trp operon genes. Such growth was accomplished by growingthe culture in a minimal medium (comprised of 6 g Na₂ HPO₄, 3 g KH₂ PO₄,0.5 g NaCl, and 1 g NH₄ Cl per liter) at 30° C. to an OD₆₀₀ of 0.3,shifting the temperature to 42° C. for 1.5 hr., and then lowering thetemperature to 30° C. for 2 hr., at which time 300 μg/mL of anthranilatewas added. The cultures were then grown another 7 hours before beingharvested and analyzed colorometrically for intracellular indole.

The colorometric indole assay for each mutant was conducted byextracting 500 μl of cells (as grown above) with 500 μl of toluene.Extraction was accomplished by vortexing the cells at room temperaturefor 5 min. The organic phase was then removed. 100 μl of the extractedorganic phase was then added to 5 ml of Assay Mixture (5.56 gp-methylaminobenzaldehyde in 1 L ethanol-acid (80 ml concentratedHCl+920 ml ethanol) ), vortexed, and allowed to stand at roomtemperature for 15-20 min., after which time the OD₅₄₀ was measured.These results were compared to a indole standard curve prepared bymeasuring the A₅₄₀ generated when 0, 2, 4, 6, 8, or 10 μg of indole(taken from a freshly prepared indole stock solution, 100 μg/ml dH₂ O)was assayed as described above.

The above procedures were used to analyze a series of mutants designedto introduce a single amino acid substitution at trpB³⁸². In addition,mutations were also generated at the position corresponding to trpB³⁷⁹.It was found that substituting Pro for the wild type residue at thisposition enabled more than five-fold increase in indole accumulation ascompared to the best trpB³⁸² mutant, namely Gly³⁸². Because two siteswere discovered to be independently capable of enabling intracellularindole accumulation, a series of double mutants, with changes at bothtrpB³⁷⁹ and trpB³⁸², were generated. The various mutants generated andthe amount of indole they produced appear in Table 1.

                  TABLE 1    ______________________________________    Mutant  Position    Substitution                                  Indole (mg/L)    ______________________________________    1       382         Asn       7    2       382         Ser       0    3       382         Ala       0    4       382         Thr       0    5       382         Gly       30    6       382         Gln       0    7       382         Arg       0    8       382         Glu       0    9       382         Phe       0    10      382         Met       18    11      379         Pro       160    12      379         Pro            382         Met       150    13      379         Gly            382         Gly       7    ______________________________________

However, none of the double routants generated showed any increasedability to accumulate indole relative to Pro³⁷⁹, although in thePro379/Met³⁸² double mutant, designated pYTrp#26, 150 mg/L indole wasdetected. This indole level was roughly the same as was detected in thebest single mutant, Pro³⁷⁹. DNA sequencing was performed on each of theindole accumulating mutants to confirm the presence of the anticipatedchanges. Because pYTrp#26 produced nearly as much indole as any singlemutant, it was chosen for further study, as a reversion to the wild typephenotype and/or genotype was much less likely to occur in a doublemutant.

EXAMPLE 3 Fermentation of Indole Accumulating Mutants

As shake flask studies indicated that substantial quantities ofintracellular indole could be produced by pYTrp#26, small scale fedbatch fermentations were conducted with this and other constructs toexamine indole production and accumulation in a more realisticindustrial setting. The fed-batch fermentations were conducted in asmall, 1 L chemostat under carbon-limited growth conditions. The initialbatch medium was prepared in a 2 L sterile bottle by combiningpreviously prepared, sterile solutions. The medium was prepared bycombining 200 ml of Solution I (6 g yeast extract plus dH₂ O to a finalvolume of 200 ml), 200 ml of Solution 2 (3.75 g (NH₄)₂ SO₄, 8.4 g K₂HPO₄, and 4.6 g KH₂ PO₄ plus dH₂ O to a final volume of 200 ml), 15 mlof a 40% glucose solution, 4.8 ml of 1M MgSO₄, 2.4 ml of a trace metalssolution (Table 2), 2.4 ml of a vitamins and minerals solution (Table3), 775 ml dH₂ O, ampicillin to 100 μg/ml, and 200 μl of antifoam.

                  TABLE 2    ______________________________________    Trace Metals Solution    Compound         g/L    ______________________________________    FeCl.sub.3.6H.sub.2 O                     27.0 ± 0.3    ZnCl.sub.2       2.0 ± 0.03    CoCl2.6H.sub.2 O 2.0 ± 0.03    NaMoO.sub.4.2H.sub.2 O                     2.0 ± 0.03    CaCl.sub.2.2H.sub.2 O                     1.0 ± 0.02    CuSO.sub.4.5H.sub.2 O                     1.9 ± 0.03    H.sub.3 BO.sub.3 0.5 ± 0.01    MnCl.sub.2.4H.sub.2 O                     1.6 ± 0.03    Sodium Citrate.2H.sub.2 O                     73.5 ± 1.0    ______________________________________

[prepare by dissolving the ingredients in about 90% of the total lotvolume with purified H20; after dissolution, adjust to the desired finallot volume using purified H₂ O; sterilize by filtration through a 0.2 μmfilter]

                  TABLE 3    ______________________________________    Vitamins and Minerals Solution    ______________________________________    Compound        g/l    ______________________________________    Biotin          0.06 ± 0.001    Folic Acid      0.04 ± 0.001    Pyridoxine      1.4 ± 0.03    Riboflavin      0.42 ± 0.008    Pantothenic Acid                    5.4 ± 0.11    Niacin          6.1 ± 0.12    ______________________________________    Compound        ml/L    ______________________________________    10 N NaOH       5.31 ± 0.11    ______________________________________

[prepare by: (a) dissolving Biotin, Folic Acid, and Riboflavin in about4% of total lot volume using purified H₂ O and 5.65±0.19% of total lotvolume of 10 N NaOH; after dissolution, adjust to 5% of total lot volumeusing purified H₂ O; (b) dissolve Pryidoxine and Niacin in about 2% oftotal lot volume using purified H₂ O and 94.2±0.19% of total lot volumeof 10 N NaOH; after dissolution, adjust to 2.5% of total lot volumeusing purified H₂ O; (c) dissolve Pantothenic Acid in about 2% of totallot volume using purified H₂ O and 0.188±0.019% of total lot volume of10 N NaOH; after dissolution, adjust to 2.5% of total lot volume usingpurified H₂ O; (d) combine the solutions prepared in (a), (b), and (c)and adjust to the total lot volume using purified H₂ O; and (e)sterilize the solution by filtration through a 0.2 μm filter]

The batch medium was then added to a previously sterilized chemostat andpreheated to 30° C. Agitation was set at 1,000 rpm, the air flow ratewas set at 3 L/min, and the pH controller was set to maintain a solutionpH of 7.0±0.2 by adding either H₃ PO₄ or NH₄ OH. The fermentor was theninoculated to a final OD₆₀₀ of about 0.02-0.03 using a fresh overnightculture. The culture was allowed to grow at 30° C. until an OD₆₀₀ ofabout 8.7 was reached. At that point, the addition of a feedsolution^(I) was initiated according to the following schedule:

    ______________________________________    OD.sub.600  Feed rate (ml/hr)    ______________________________________    8.7         1.25    14.9        1.9    19.8        3.1    24.8        5.0    37.2        7.5    62.0        11.25    74.0        20.0    86.8        25.0    ______________________________________

When the culture reached an OD₆₀₀ of 65 to 75, transcription of themodified trp operon was induced by shifting the culture temperature to42° C. for 1.5 hr. After induction, the culture's temperature wasquickly adjusted down to 30° C. The fermentation was then continued foranother 8 hr.

Using the above fermentation procedure, indole synthesis andaccumulation was studied in pYTrp#26. The results appear in FIG. 2. Inaddition, mutant #5 (Table 1, supra), which harbored only a singlemutation and was an intermediate indole producer in shake flasks, wasalso tested in the 1 L fermentor. The results of these fermentationsappear in FIG. 3. As shown, pYTrp#26 produced approximately 1 g/L ofindole 13 hours after trp operon expression had been induced. Theability of the cells to grow in the presence of more than 0.04-0.05%indole was also unexpected in view of the prior art. For example, seeBang et al., supra, (1983). Optimumization of fermentation conditionsshould enable significantly increased levels of indole production.

EXAMPLE 4 Deletion of the Trp Operon Promoter

In addition to generating specific mutations in particular genes of thetrp operon designed to enable high level indole production, the indoleproduction pathway can be further refined through the deletion of theendogenous trp promoter. Removal of this promoter should increase theefficiency of transcription of the operon from the P_(L) promoter byeliminating the potential for repression by the trp repressor protein.In addition, removal of the trp promoter will enable improvedtranscriptional regulation of the trp operon employed in the practice ofthe present invention, namely by reducing "leakiness," as the trppromoter will no longer enable transcription initiation even while P_(L)is repressed.

Removal of the endogenous trp promoter was accomplished by digestingpYTrp#26 with Xba I and Spe I, the Xba I site being in the plasmid'spolylinker, and the Spe I site residing near the 3' end of the trppromoter. Removal of this fragment also served to remove about 400 bp ofextraneous DNA 5' of the trp promoter. Also, because Xba I and Spe Ileave identical 3' overhangs following digestion, removal of theintervening fragment allows the complementary "sticky ends" to cometogether. Thus, following agarose gel purification, the sticky ends ofthe linearized plasmid were allowed to anneal and the sequences ligated.In the resultant construct, both the Xba I and Spe I sites were lost.This new trp promoterless construct was designated pYTrp#26p-.

Shake flask experiments were then conducted to compare indole productionin pYTrp#26p- with that in pYTrp#26. These studies indicated thatpYTrp#26p-made as much or slightly more indole, and appeared to bebetter regulated, than pYTrp#26.

To substantiate the shake flask results and to make a comparison in amore commercially realistic setting, 1 L fermentations were conductedwherein both constructs were tested for indole production. Thefermentation conditions used here were the same as those used in Example2. These results indicated that the new promoterless trp operonconstruct not only produced more indole than pYTrp#26, but that thepromoterless construct also exhibited improved regulation of trp operonexpression. Prior to temperature induction of the P_(L) promoter,pYTrp#26p-produced little or no indole. In contrast, the pYTrp#26construct made about 30% of its total indole yield prior to trp operoninduction. This improved regulation also appeared to increase the growthrate of pYTrp#26p- as compared to pYTrp#26.

EXAMPLE 5 Deletion of the Trp Operon Attenuator

As described in Example 4, deletion of endogenous trp regulatory regionsfrom the trp operon utilized in this invention can result is increasedindole synthesis. Beyond removal of the trp promoter, it was alsopossible to generate a useful modified trp operon that had the trpattenuator region deleted as well. As the attenuator can be responsiblefor up to 90% of the transcriptional repression of the trp operon invivo, removal of this region was expected to enable an improved indoleproduction rate.

To delete the trp attenuator, which was located between the trp promoterand amino terminus of the trpE gene, site directed mutagenesis wasconducted wherein a unique Xho I site was added nine codons downstreamfrom the trpE gene initiation codon. Addition of this restriction siteenabled the maintenance the wild type amino acid sequence of the trpEgene, thanks to the degenerate nature of the genetic code. Using thissite, it was possible to remove the native promoter/attenuator region bydigesting with Xho I and Xba I. A synthetic Xho I/Xba I linker, designedto reconstitute the nine 5' codons of the trpE gene, was then employedto complete the construct. In addition, the linker was designed tocontain an efficient ribosomal binding site with consensus spacing fromthe P_(L). Finally, the 3' end of the linker was engineered to containthe initial nine codons for the trpE gene, and the codons used are those"preferred" by E. coli. Thus, the result of this construction,designated pYTrp#26att-, was to delete the trp promoter and attenuatorregions; position the trpE gene close to the PL, separated by a strongribosomal binding site; and to provide a trpE gene with "preferred" E.coli codons in the first nine positions of the gene's open readingframe.

This plasmid was then transformed into FM5 and compared in shake flasksto FM5 harboring pYTrp#26p-. The pYTrp#26att- harboring strain was foundto grow more slowly than the strain transformed with pYTrp#26p-.However, the attenuator deficient construction enabled the production ofconsiderably more indole.

EXAMPLE 6 Deletion of the Trp Operon Rho-dependent Terminator

In addition to removing the trp promoter, attenuator (or both) andextraneous, non-coding 5' DNA from the trp operon, it is also possibleto delete DNA 3' to the trpA gene. Along these lines, a DNA sequenceapproximately 250 bp in length containing a rho-dependent terminator wasremoved from pYTrp#26 by digesting the plasmid with Ssp I and Barn HIusing a 5' exonuclease activity to remove the 5' overhang left by theBam HI digestion; purifying the linearized plasmid from the small,excised fragment; and ligating the resultant gel-purified, linearizedplasmid to itself. When this construction, which still contains arho-independent terminator 3' to the trpA gene, was compared for indoleproduction in shake flasks against pYTrp#26, no improvement wasobserved. However, deletion of this extraneous DNA apparently had nodeleterious effects on indole production or plasmid stability, and thusthe deletion may be useful in that the indole-producing trp operon wasfurther streamlined through the elimination of extraneous noncoding DNA.

EXAMPLE 7 Translocated Host Strains

Because biosynthetic indigo production from glucose requires both anenzymatic pathway having the ability to produce intracellular indole andan enzymatic pathway possessing the ability to convert that indole toindoxyl, it is necessary that the strain which produces indigo harborboth pathways. One way in which this may be accomplished is to integrateeither of the two pathways (trp or NDO) into the chromosome of anappropriate host bacterium and, after successful integration, transformthe host with the other pathway such that it will be maintainedextrachromosomally.

In one such method, the indole-generating trp operon was excised frompYTrp#26 as an Aat I to Bam HI fragment, purified through an agarose geland then inserted into the translocation vector pCFM2202, which containsa DNA fragment from a pBR322 construct comprising Tn5 transposase geneand including the IS50L insertion sequence essential for chromosomalintegration on either side of the DNA to be integrated [Sasakawa et al.,(1982) Proc. Natl. Acad. Sci., USA, vol. 79, pp. 7450-7454]. Thetranslocation vector provides for selection using the antibiotictetracycline. In addition, it directs the integration of the desired,inserted DNA sequence, here the modified trp operon, in conjunction withthe structural gene for the cI₈₅₇ regulatory element, which itself isunder the control of a low-level constitutive promoter. Followingassembly, the trp operon integration plasmid was designated pCFM2202Trpand was transformed into E. coli strain FM5. Following transformation,the strain was redesignated DM2.

After transformation, several transformants were selected and passagedfor 13 generations in nonselective media in order to "cure" the cells ofthe plasmid. Subsequent to passaging, plasmid deficient cells containingthe a trp operon were identified by colony hybridization [Manniatas etal., supra] using an oligonucleotide probe specific for the #26 trpBgene mutation. Several isolates were examined for their indole producingability. The translocated strain designated DM2#26 was found to be themost proficient indole producer of the translocatants. DM2#26 was thencompared against FM5 transformed with pYTrp#26. Upon induction, thetranslocated strain was found to produce approximately 20% more indolethan its plasmid bearing sibling, although the integrant grew moreslowly than the strain harboring the extrachromosomal element. Finally,in an effort to assess whether or not DM2#26 could produce indigo ifpresented with an appropriate indole to indoxyl conversion mechanism, itwas transformed with pFd911ABC. A resultant transformant, harboring theintegrated modified trp operon and a plasmid-borne NDO pathway, wasobserved to make low levels of indigo in shake flasks.

While the present invention has been described in terms of preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art in light of the above description.Therefore, it is intended that the appended claims cover all suchvariations which come within the scope of the invention as claimed.

    __________________________________________________________________________       SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii ) NUMBER OF SEQUENCES: 5    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 12 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CATCGAT TCTAG       12    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    AATTCTA GAATCGAT GACGT      20    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 36 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GTTAAC CTTTCC GGTCGCG GCGATAA AGACATC TTC   36    ValAsn LeuSer GlyArg GlyAsp LysAsp IlePhe    1  5  10    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1193 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: unknown     (D) TOPOLOGY: unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    ATGACA ACATTA CTTAACC CCTATT TTGGTGA GTTTGGC GGCATG TAC 48    MetThr ThrLeu LeuAsn ProTyr PheGly GluPhe GlyGly MetTyr    1  5  10  15    GTGCCA CAAATC CTGATG CCTGCTC TGCGCCA GCTGGAA GAAGCT TTT 96    ValPro GlnIle LeuMet ProAla LeuArg GlnLeu GluGlu AlaPhe     20  2 5  30    GTCAGT GCGCAA AAAGATC CTGAATT TCAGGCT CAGTTC AACGACC TG1 44    ValSer AlaGln LysAsp ProGlu PheGln AlaGln PheAsn AspLeu     35  40  45    CTGAAA AACTAT GCCGGGC GTCCAAC CGCGCTG ACCAAA TGCCAGA AC1 92    LeuLys AsnTyr AlaGly ArgPro ThrAla LeuThr LysCys GlnAsn    50   55  60    ATTACA GCCGGG ACGAACA CCACGCT GTATCT CAAGCGT GAAGAT TTG 240    IleThr AlaGly ThrAsn ThrThr LeuTyr LeuLys ArgGlu AspLeu    65  70  75   80    CTGCAC GGCGGC GCGCATA AAACTAA CCAGGTG CTGGGG CAGGCGT TG2 88    LeuHis GlyGly AlaHis LysThr AsnGln ValLeu GlyGln AlaLeu      85  90  95    CTGGCG AAGCGG ATGGGTA AAACCGA AATCAT CGCCGAA ACCGGT GCC 336    LeuAla LysArg MetGly LysThr GluIle IleAla GluThr GlyAla     100  10 5  110    GGTCAG CATGGC GTGGCGT CGGCCCT GGCCAGC GCCCTG CTCGGCC TG3 84    GlyGln HisGly ValAla SerAla LeuAla SerAla LeuLeu GlyLeu     115  120  125    AAATGC CGTATT TATATGG GTGCCAA AGACGTT GAACGC CAGTCGC CT4 32    LysCys ArgIle TyrMet GlyAla LysAsp ValGlu ArgGln SerPro    130  1 35  140    AACGTT TTTCGT ATGCGCT TAATGGG TGCGGAA GTGATC CCGGTGC AT4 80    AsnVal PheArg MetArg LeuMet GlyAla GluVal IlePro ValHis    145  150  155  1 60    AGCGGT TCCGCG ACGCTGA AAGATGC CTGTAAC GAGGCG CTGCGCG AC5 28    SerGly SerAla ThrLeu LysAsp AlaCys AsnGlu AlaLeu ArgAsp      165  170  175    TGGTCC GGTAGT TACGAAA CCGCGCA CTATAT GCTGGGC ACCGCA GCT 576    TrpSer GlySer TyrGlu ThrAla HisTyr MetLeu GlyThr AlaAla     180  18 5  190    GGCCCG CATCCT TATCCGA CCATTG TGCGTGA GTTTCAG CGGATG ATT 624    GlyPro HisPro TyrPro ThrIle ValArg GluPhe GlnArg MetIle     195  200  205    GGCGAA GAAACC AAAGCGC AGATTCT GGAAAGA GAAGGT CGCCTGC CG6 72    GlyGlu GluThr LysAla GlnIle LeuGlu ArgGlu GlyArg LeuPro    210  2 15  220    GATGCC GTTATC GCCTGTG TTGGCGG CGGTTCG AATGCC ATCGGCA TG7 20    AspAla ValIle AlaCys ValGly GlyGly SerAsn AlaIle GlyMet    225  230  235  2 40    TTTGCT GATTTC ATCAATG AAACCAA CGTCGGC CTGATT GGTGTGG AG7 68    PheAla AspPhe IleAsn GluThr AsnVal GlyLeu IleGly ValGlu      245  250  255    CCAGGT GGTCAC GGTATCG AAACTGG CGAGCAC GGCGCA CCGCTAA AA8 16    ProGly GlyHis GlyIle GluThr GlyGlu HisGly AlaPro LeuLys     260  26 5  270    CATGGT GCGGTG GGTATCT ATTTCGG TATGAAA GCGCCG ATGATGC AA8 64    HisGly AlaVal GlyIle TyrPhe GlyMet LysAla ProMet MetGln     275  280  285    ACCGAA GACGGG CAGATTG AAGAATC TTACTCC ATCTCC GCCGGAC TG9 12    ThrGlu AspGly GlnIle GluGlu SerTyr SerIle SerAla GlyLeu    290  2 95  300    GATTTC CCGTCT GTCGGCC CACAACA CGCGTAT CTTAAC AGCACTG GA9 60    AspPhe ProSer ValGly ProGln HisAla TyrLeu AsnSer ThrGly    305  310  315  3 20    CGCGCT GATTAC GTGTCT ATTACCG ATGATGA AGCCCTT GAAGCC TTC1 008    ArgAla AspTyr ValSer IleThr AspAsp GluAla LeuGlu AlaPhe      325  330  335    AAAACG CTGTGC CTGCACG AAGGGAT CATCCCG GCGCTG GAATCCT CC10 56    LysThr LeuCys LeuHis GluGly IleIle ProAla LeuGlu SerSer     340  34 5  350    CACGCC TTGGCC CATGCGT TGAAAAT GATGCGC GAAAAC CCGGATA AA11 04    HisAla LeuAla HisAla LeuLys MetMet ArgGlu AsnPro AspLys     355  360  365    GAGCAG CTACTG GTGGTTA ACCTTTC CGGTCGC GGCGAT AAAGACA TC11 52    GluGln LeuLeu ValVal AsnLeu SerGly ArgGly AspLys AspIle    370  3 75  380    TTCACC GTTCAC GATATTT TGAAAGC ACGAGGG GAAATC TG 11 93    PheThr ValHis AspIle LeuLys AlaArg GlyGlu Ile    385  390  395    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 397 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    MetThr ThrLeu LeuAsn ProTyr PheGly GluPhe GlyGly MetTyr    1  5  10  15    ValPro GlnIle LeuMet ProAla LeuArg GlnLeu GluGlu AlaPhe     20  2 5  30    ValSer AlaGln LysAsp ProGlu PheGln AlaGln PheAsn AspLeu     35  40  45    LeuLys AsnTyr AlaGly ArgPro ThrAla LeuThr LysCys GlnAsn    50   55  60    IleThr AlaGly ThrAsn ThrThr LeuTyr LeuLys ArgGlu AspLeu    65  70  75   80    LeuHis GlyGly AlaHis LysThr AsnGln ValLeu GlyGln AlaLeu      85  90  95    LeuAla LysArg MetGly LysThr GluIle IleAla GluThr GlyAla     100  10 5  110    GlyGln HisGly ValAla SerAla LeuAla SerAla LeuLeu GlyLeu     115  120  125    LysCys ArgIle TyrMet GlyAla LysAsp ValGlu ArgGln SerPro    130  1 35  140    AsnVal PheArg MetArg LeuMet GlyAla GluVal IlePro ValHis    145  150  155  1 60    SerGly SerAla ThrLeu LysAsp AlaCys AsnGlu AlaLeu ArgAsp      165  170  175    TrpSer GlySer TyrGlu ThrAla HisTyr MetLeu GlyThr AlaAla     180  18 5  190    GlyPro HisPro TyrPro ThrIle ValArg GluPhe GlnArg MetIle     195  200  205    GlyGlu GluThr LysAla GlnIle LeuGlu ArgGlu GlyArg LeuPro    210  2 15  220    AspAla ValIle AlaCys ValGly GlyGly SerAsn AlaIle GlyMet    225  230  235  2 40    PheAla AspPhe IleAsn GluThr AsnVal GlyLeu IleGly ValGlu      245  250  255    ProGly GlyHis GlyIle GluThr GlyGlu HisGly AlaPro LeuLys     260  26 5  270    HisGly AlaVal GlyIle TyrPhe GlyMet LysAla ProMet MetGln     275  280  285    ThrGlu AspGly GlnIle GluGlu SerTyr SerIle SerAla GlyLeu    290  2 95  300    AspPhe ProSer ValGly ProGln HisAla TyrLeu AsnSer ThrGly    305  310  315  3 20    ArgAla AspTyr ValSer IleThr AspAsp GluAla LeuGlu AlaPhe      325  330  335    LysThr LeuCys LeuHis GluGly IleIle ProAla LeuGlu SerSer     340  34 5  350    HisAla LeuAla HisAla LeuLys MetMet ArgGlu AsnPro AspLys     355  360  365    GluGln LeuLeu ValVal AsnLeu SerGly ArgGly AspLys AspIle    370  3 75  380    PheThr ValHis AspIle LeuLys AlaArg GlyGlu Ile    385  390  395

What is claimed is:
 1. A non-naturally occuring tryptophan synthase beta-polypeptide comprising an amino acid residue selected from the group consisting of Pro, Val, Ile, Leu, and Ala, at amino acid position trpB³⁷⁹ and comprising an amino acid residue selected from the group consisting of Asn, Gly, and Met at amino acid position trpB³⁸² which when incorporated into tryptophan synthase results in enhanced indole accumulation relative to a tryptophan synthase beta subunit having Asn instead of Lys at amino acid position
 382. 2. A tryptophan synthase beta-subunit polypeptide according to claim 1 which comprises Pro at amino acid position trpB³⁷⁹.
 3. A tryptophan synthase beta-subunit polypeptide according to claim 1 which comprises an amino acid residue selected from the group consisting of Gly and Met at amino acid position trpB³⁸².
 4. A tryptophan synthase beta-subunit polypeptide according to claim 2 which comprises Met at amino acid position trpB³⁸².
 5. A tryptophan synthase beta-subunit polypeptide according to claim 1 comprising Pro at amino acid position trpB³⁷⁹ and comprising Met at amino acid position trpB³⁸². 